Biofilm

Biofilms have been found in a wide variety of microbial wound infections accounting for nearly 80% of all such infections. It has also been estimated that biofilm is present within 65% of nosocomial infections and that the annual cost of treating these infections is greater than $1 billion in the United States. Electron microscopes have revealed that 60% of biopsies from chronic wounds contain biofilm as opposed to 6% of biopsies from acute wounds. Since biofilms are a major contributor to chronic inflammatory diseases, it is likely that almost all chronic wounds have biofilm communities within the wound bed. It is thought that biofilms may impair cutaneous wound healing and reduce the effectiveness of topical antibiotics and thereby delay or inhibit the healing or treatment of infected wounds.

Frequently Asked Questions

What is Biofilm?

Biofilm is any group of microorganisms in which cells stick to each other on a surface. These cells embed in an adherent protective matrix of extracellular polymeric substance (EPS). Biofilm EPS is a polymeric, composed of extracellular DNA, proteins, nucleic acids and polysaccharides. The EPS is more or less strongly hydrated. The microbial cells within a biofilm are different than planktonic cells (free floating and dispersed) of the same organism. In contrast, a biofilm is a fixed substrate that can adapt internally to different conditions by its host microbes. Biofilms are dynamic heterogeneous communities that are continuously changing. The EPS matrix has increased resistance to antibiotics and antiseptics, in some cases 1,000 fold. It is in this protective matrix that lateral transfer of genes takes place, resulting in a more stable biofilm structure.

How does biofilm form?

Formation begins with the attachment of free-floating (planktonic) microorganisms to the surface of the wound bed. The first cells to attach are typically weak and at this stage the adhesion may be reversible. If the microbes are not immediately separated from the wound bed surface, they will adhere themselves permanently (sessile). Those microbes with increased hydrophobicity, have reduced repulsion between the extracellular matrix and the bacterium, increasing resistance and adhesion. Once colonization begins, the biofilm grows through cell division and cellular recruitment. The polysaccharide matrix encloses the biofilm creating a semi-permeable wall. The final stage of biofilm formation is called dispersion. In this stage, the biofilm that has been established may change in size and shape, enabling it to spread to other surfaces. As the microbes differentiate, they change gene patterns in ways that promote their own survival.

What are the characteristics of biofilm?

Biofilms can quickly grow to become macroscopic (visible to the naked eye). They may contain many different types of organisms (polymicrobial) including bacteria, fungi and yeast, most of which are thought to be present on the skin surface of infected wounds. Research has shown that early inhabitants of infected wounds are generally found in the skin flora, mostly gram-positive bacteria. These bacteria use whatever oxygen is available to them and provide growth factors for anaerobes to establish and flourish within the biofilm, giving way to gram-negative bacteria. By day 6, gram-negative anaerobes become more prominent. It is likely that biofilms play a prominent role in this progression and as a result have a pronounced effect on inflammation, infection and delayed healing.

How quickly do biofilms form?

Biofilms may attach within minutes, and form strongly attached communities within 2 to 4 hours. The EPS becomes increasingly tolerant to biocides, antibiotics and antiseptics within 6 to 12 hours. The colony can begin to shed planktonic bacterium within 2 to 4 days. Once disturbed, biofilm can recover and reform to maturity within 24 hours.

Why are wounds infected with biofilm so hard to treat?

First, biofilm bacteria are less susceptible to our immune system, so they can persist for a long time. The phagocytes (part of our natural immunity) have difficulty in ingesting these bacteria as there are no specific antibodies to trigger an antigenic response. If antibodies are present, the EPS renders them ineffective. Not only are host defenses unable to effectively combat biofilm, but their persistence causes progressive tissue damage. When the polymorphonucleocytes (PMNs) are not able to digest the bacterium in the biofilm the PMNs release large amounts of toxic cytokines, leading to chronic inflammation and destruction of nearby tissue. Second, biofilm also displays innate resistance to antimicrobials, which are either rapidly inactivated or fail to penetrate into the biofilm. Bacteria within biofilms may be 1,000 times more resistant than the same bacteria in a planktonic state. Finally, biofilms increase the gene transfer between and among bacteria, which leads to increasing resistance to antibiotics by transferring genes for resistance to neighboring, more susceptible bacteria.

Biofilm expresses resistance to antibiotics in part due to a reduced growth rate, so called quiescent bacteria or the equivalent of hibernation. Because bacteria need to be metabolically active in order for antibiotics to be effective, hibernating bacteria are unaffected by antibiotics that would normally kill active bacteria. Research has shown that the lowest concentration required to kill or eliminate bacterial biofilm far exceeds the maximum amount that is non-harmful to healthy tissue.

What role do biofilms play in delaying wound healing?

Biofilms stimulate a chronic inflammatory response in order to rid the wound of the biofilm. This results in neutrophils and macrophages flooding the wound amphitheater. Although they quickly become overwhelmed, the inflammatory cells secrete high levels of proteases and reactive oxygen species (ROS), sometimes referred to as oxygen bursts. The proteases break down the attachments between the biofilm and the tissues at the bed of the wound and the neutrophils lyse the bacteria in the biofilm, effectively, killing the bacteria. However, the ROS and the proteases also damage normal and healing tissues and impair the healing process. By inducing an inflammatory response, the biofilm protects the microorganisms it harbors and increases exudate production, which serves as a source of nutrition to help perpetuate the biofilm.

How can biofilm burden be reduced?

Debridement and wound cleansing in addition to the removal of exudate and decreasing edema within the wound are effective measures for reducing biofilm burden. Pure hypochlorous solution (PhaseOne) has been shown in laboratory studies to have less than a 60-second kill time in solution with no known resistance and to be able to neutralize the resultant endotoxins that infiltrate the wound once the extracellular walls have been ruptured, thus preventing damage to underlying new tissue growth, including fibroblasts. Hypochlorous solution is non-toxic even in high concentrations (3ppm) and mimics the oxygen bursts that are an innate defense mechanism within our body’s neutrophils. Applying negative pressure wound therapy (NPWT) in combination with hypochlorous solution in a chronic wound will remove exudate from the wound and reduce edema, while drawing the margins of the wound (peri-wound) closer together with continuous and intermittent negative pressure. Antimicrobial contact layers that are able to mechanically attract hydrophobic bacteria such as Cutimed Sorbact can be used to physically remove biofilm and planktonic pathogens from the wound site without cytolysis of the infecting pathogen.